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PubMed2003

Interaction of the tyrosine kinase Pyk2 with the N-methyl-D-aspartate receptor complex via the Src homology 3 domains of PSD-95 and SAP102.

The protein-tyrosine kinase Pyk2/CAKbeta/CADTK is a key activator of Src in many cells. At hippocampal synapses, induction of long term potentiation requires the Pyk2/Src signaling pathway, which up-regulates the activity of N-methyl-d-aspartate-type glutamate receptors. Because localization of protein kinases close to their substrates is crucial for effective phosphorylation, we investigated how Pyk2 might be recruited to the N-methyl-d-aspartate receptor complex. This interaction is mediated by PSD-95 and its homolog SAP102. Both proteins colocalize with Pyk2 at postsynaptic dendritic spines in the cerebral cortex. The proline-rich regions in the C-terminal half of Pyk2 bind to the SH3 domain of PSD-95 and SAP102. The SH3 and guanylate kinase homology (GK) domain of PSD-95 and SAP102 interact intramolecularly, but the physiological significance of this interaction has been unclear. We show that Pyk2 effectively binds to the Src homology 3 (SH3) domain of SAP102 only when the GK domain is removed from the SH3 domain. Characterization of PSD-95 and SAP102 as adaptor proteins for Pyk2 fills a critical gap in the understanding of the spatial organization of the Pyk2-Src signaling pathway at the postsynaptic site and reveals a physiological function of the intramolecular SH3-GK domain interaction in SAP102.

Animals↗
PubMed2002

Dynamin is a minibrain kinase/dual specificity Yak1-related kinase 1A substrate.

The minibrain kinase (Mnbk)/dual specificity Yak 1-related kinase 1A (Dyrk1A) gene is implicated in the mental retardation associated with Down's syndrome. It encodes a proline-directed serine/threonine kinase whose function has yet to be defined. We have used a solid-phase Mnbk/Dyrk1A kinase assay to aid in the search for the cellular Mnbk/Dyrk1A substrates. The assay revealed that rat brain contains two cytosolic proteins, one with a molecular mass of 100 kDa and one with a molecular mass of 140 kDa, that were prominently phosphorylated by Mnbk/Dyrk1A. The 100-kDa protein was purified and identified as dynamin 1. The conclusion was further supported by evidence that a recombinant glutathione S-transferase fusion protein containing dynamin isoform 1aa was phosphorylated by Mnbk/Dyrk1A. In addition to isoform 1aa, Mnbk/Dyrk1A also phosphorylated isoforms 1ab and 2aa but not human MxA protein when analyzed by the solid-phase kinase assay. Upon Mnbk/Dyrk1A phosphorylation, the interaction of dynamin 1 with the Src homology 3 domain of amphiphysin 1 was reduced. However, when Mnbk/Dyrk1A phosphorylation was allowed to proceed more extensively, the phosphorylation enhanced rather than reduced the binding of dynamin 1 to amphiphysin 1. The result suggests that Mnbk/Dyrk1A can play a dual role in regulating the interaction of dynamin 1 with amphiphysin 1. Mnbk/Dyrk1A phosphorylation also reduced the interaction of dynamin with endophilin 1, whereas the same phosphorylation enhanced the binding of dynamin 1 to Grb2. Nevertheless, the dual function of Mnbk/Dyrk1A phosphorylation was not observed for the interaction of dynamin 1 with endophilin 1 or Grb2. The interactions of dynamin with amphiphysin and endophilin are essential for the formation of endocytic complexes; our results suggest that Mnbk/Dyrk1A may function as a regulator controlling the assembly of endocytic apparatus.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Epidermal growth factor receptor targeting prevents uncoupling of the Grb2-SOS complex.

Insulin stimulates the Ras/Raf/MEK/ERK pathway leading to feedback phosphorylation of the Ras guanylnucleotide exchange protein SOS and dissociation of Grb2 from SOS. Even though epidermal growth factor (EGF) also stimulates ERK activity and phosphorylation of SOS similar to insulin, EGF induces a dissociation of the Grb2-SOS complex from Shc. To determine the molecular basis for this difference, we examined the signaling properties of a mutant EGF receptor lacking the five major autophosphorylation sites. Although EGF stimulation of the mutant EGF receptor activates ERK and phosphorylation of both Shc and SOS, it fails to directly associate with either Shc or Grb2. However, under these conditions EGF induces a dissociation of the Grb2-SOS complex suggesting a role for receptor and/or plasma membrane targeting in the stabilization of Grb2-SOS interaction. Consistent with this hypothesis, expression of an SH2 domain Grb2 mutant which is unable to mediate plasma membrane targeting of the Grb2-SOS complex results in both insulin- and EGF-stimulated uncoupling of Grb2 from SOS. Furthermore, a plasma membrane-bound Grb2 fusion protein remains constitutively associated with SOS. Together, these data demonstrate that EGF stimulation prevents the feedback uncoupling of Grb2 from SOS by inducing a persistent plasma membrane receptor targeting of the Grb2-SOS complex.

Adaptor Proteins, Signal Transducing↗
PubMed1996

p210BCR/ABL induces formation of complexes containing focal adhesion proteins and the protooncogene product p120c-Cbl.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder caused by the t(9;22) translocation. This translocation creates a unique tyrosine kinase oncogene, bcr/abl, whose product, p210BCR/ABL, is localized to the actin cytoskeleton. One of the major tyrosine phosphoproteins in cells transformed by p210BCR/ABL is the protooncoprotein p120c-Cbl. We have previously shown that p210BCR/ABL induces formation of a multimeric complex of proteins which include p120c-Cbl, phosphotidylinositol-3' kinase, and p210BCR/ABL itself. Here we show that certain focal adhesion proteins are also part of this complex, including paxillin and talin. The sites in paxillin required to bind to p120c-Cbl in this complex have been partially mapped. The interaction of pl20c-Cbl with paxillin is specific, since other focal adhesion proteins, such as p125FAK, vinculin, and alpha-actinin, are not in this complex. The binding of p120c-Cbl to the focal adhesion protein paxillin could contribute to the known adhesive defects of CML cells.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Cloning of a novel signal-transducing adaptor molecule containing an SH3 domain and ITAM.

We molecularly cloned a cDNA coding for a novel phosphotyrosine molecule with a 70 kDa molecular mass, named STAM (signal transducing adaptor molecule), which is tyrosine-phosphorylated rapidly after stimulation with various cytokines such as IL-2, IL-3, IL-4, IL-7, GM-CSF, EGF and PDGF. STAM contains an SH3 (Src-homology 3) domain and the ITAM (immunoreceptor tyrosine-based activation motif), suggesting that STAM acts as an adaptor molecule involved in signal transducing pathways from the cytokine receptors.

Adaptor Proteins, Signal Transducing↗
PubMed2003

The role of the Ca2+-sensitive tyrosine kinase Pyk2 and Src in thrombin signalling in rat astrocytes.

We have recently demonstrated that multiple signalling pathways are involved in thrombin-induced proliferation in rat astrocytes. Thrombin acts by protease-activated receptor-1 (PAR-1) via mitogen-activated protein kinase activity. Signalling includes both Gi/(betagamma subunits)-phosphatidylinositol 3-kinase and a Gq-phospholipase C/Ca2+/protein kinase C (PKC) pathway. In the present study, we investigated the possible protein tyrosine kinases which might be involved in thrombin signalling cascades. We found that, in astrocytes, thrombin can evoke phosphorylation of proline-rich tyrosine kinase (Pyk2) via PAR-1. This process is dependent on the increase in intracellular Ca2+ and PKC activity. Moreover, in response to thrombin stimulation Pyk2 formed a complex with Src tyrosine kinase and adapter protein growth factor receptor-bound protein 2 (Grb2), which could be coprecipitated. Furthermore, both thrombin-induced Pyk2 phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation can be attenuated by Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. From these data we conclude that PAR-1 uses Ca2+- and PKC-dependent Pyk2 to activate Src, thereby leading to ERK1/2 activation, which predominantly recruits Grb2 in rat astrocytes.

Adaptor Proteins, Signal Transducing↗
PubMed1993

A biosensor approach to probe the structure and function of the p85 alpha subunit of the phosphatidylinositol 3-kinase complex.

Phosphatidylinositol 3-kinase, which generates putative novel second messenger phospholipids, is a heterodimer composed of regulatory adaptor 85-kDa and catalytic 110-kDa subunits. The p85 alpha subunit contains a NH2-terminal src homology (SH) 3 domain, a region with homology to the product of the breakpoint cluster region (bcr) gene, and a COOH-terminal portion of the molecule which contains two SH2 domains, separated by a spacer region. In this study a panel of monoclonal antibodies (mAb) was raised against recombinant bovine p85 alpha to probe its multidomain structure in relation to function. These mAbs were characterized using a BIAcore biosensor instrument. Epitopes for nine mAbs were mapped in relation to the domain structure of p85 alpha using recombinant protein fragments expressed in bacteria. These mAbs were then used to map the sites on p85 alpha which are involved in growth factor receptor binding. Two interesting classes of functional mAbs were identified. First, mAb U14, whose epitope lies within the NH2-terminal SH2 domain of p85 alpha, blocked the interaction of p85 alpha with activated protein-tyrosine kinase receptors. Second, real-time binding experiments using phospholipid-containing vesicles showed that p85 alpha by itself could specifically bind certain phospholipids. Two mAbs (U9 and U15) with epitopes located in the inter-SH2 spacer region blocked the binding of lipids to this site. The relevance of these observations to understanding the relationship of structure to function of p85 and the phosphatidylinositol 3-kinase are discussed.

Amino Acid Sequence↗
PubMed2004

Regulation of blood-testis barrier dynamics: an in vivo study.

An in vivo model was used to investigate the regulation of tight junction (TJ) dynamics in the testis when adult rats were treated with CdCl(2). It was shown that the CdCl(2)-induced disruption of the blood-testis barrier (BTB) associated with a transient induction in testicular TGF-beta2 and TGF-beta3 (but not TGF-beta1) and the phosphorylated p38 mitogen activated protein (MAP) kinase, concomitant with a loss of occludin and zonula occludens-1 (ZO-1) from the BTB site in the seminiferous epithelium. These results suggest that BTB dynamics in vivo are regulated by TGF-beta2/-beta3 via the p38 MAP kinase pathway. Indeed, SB202190, a specific p38 MAP kinase inhibitor, blocked the CdCl(2)-induced occludin and ZO-1 loss from the BTB. This result clearly illustrates that CdCl(2) mediates its BTB disruptive effects via the TGF-beta3/p38 MAP kinase signaling pathway. Besides, this CdCl(2)-induced occludin and ZO-1 loss from the BTB also associated with a significant loss of the cadherin/catenin and the nectin/afadin protein complexes at the site of cell-cell actin-based adherens junctions (AJs). An induction of alpha(2)-macroglobulin (a non-specific protease inhibitor) was also observed during BTB damage and when the seminiferous epithelium was being depleted of germ cells. These data illustrate that a primary disruption of the BTB can lead to a secondary loss of cell adhesion function at the site of AJs, concomitant with an induction in protease inhibitor, which apparently is used to protect the epithelium from unwanted proteolysis. alpha(2)-Macroglobulin was also shown to associate physically with TGF-beta3, afadin and nectin 3, but not occludin, E-cadherin or N-cadherin, indicating its possible role in junction restructuring in vivo. Additionally, the use of SB202190 to block the TGF-beta3/p-38 MAP kinase pathway also prevented the CdCl(2)-induced loss of cadherin/catenin and nectin/afadin protein complexes from the AJ sites, yet it had no apparent effect on alpha(2)-macroglobulin. These results demonstrate for the first time that the TGF-beta3/p38 MAP kinase signaling pathway is being used to regulate both TJ and AJ dynamics in the testis, mediated by the effects of TGF-beta3 on TJ- and AJ-integral membrane proteins and adaptors, but not protease inhibitors.

Adherens Junctions↗
PubMed1994

Insulin receptor substrate-1 (IRS1) and Shc compete for a limited pool of Grb2 in mediating insulin downstream signaling.

Expression of the insulin receptor substrate-1 (IRS1) or Shc cDNA resulted in both increased protein and insulin-stimulated tyrosine phosphorylation of IRS1 and Shc proteins, respectively. Although expression of Shc had no significant effect on insulin-stimulated mitogen-activated protein (MAP) kinase gel shift or c-fos transcriptional activation, expression of IRS1 inhibited these responses. The effect of IRS1 expression on the formation of multisubunit signaling complexes was determined by a series of indirect co-immunoprecipitations. Grb2 immunoprecipitation from IRS1-transfected and insulin-treated cells demonstrated an increased coimmunoprecipitation of Syp and the p85 regulatory subunit of the phosphatidylinositol 3-kinase. Similarly, cell extracts immunoprecipitated with a p85 antibody displayed an increased co-immunoprecipitation of Syp and Grb2. However, expression of IRS1 increased the extent of Grb2 associated with IRS1 with a concomitant reduction in the amount of Grb2 associated with Shc. Furthermore, increased expression of Shc reduced the amount of Grb2 bound to IRS1 with a concomitant increase in Grb2 associated with Shc. Together, these data demonstrate that IRS1 and Shc compete for a limited cellular pool of Grb2, and insulin activation of MAP kinase and c-fos transcription predominantly occur through the Shc-Grb2 signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Identification of an expanded binding surface on the FADD death domain responsible for interaction with CD95/Fas.

The initiation of programmed cell death at CD95 (Fas, Apo-1) is achieved by forming a death-inducing signaling complex (DISC) at the cytoplasmic membrane surface. Assembly of the DISC has been proposed to occur via homotypic interactions between the death domain (DD) of FADD and the cytoplasmic domain of CD95. Previous analysis of the FADD/CD95 interaction led to the identification of a putative CD95 binding surface within FADD DD formed by alpha helices 2 and 3. More detailed analysis of the CD95/FADD DD interaction now demonstrates that a bimodal surface exists in the FADD DD for interaction with CD95. An expansive surface on one side of the domain is composed of elements in alpha helices 1, 2, 3, 5, and 6. This major surface is common to many proteins harboring this motif, whether or not they are associated with programmed cell death. A secondary surface resides on the opposite face of the domain and involves residues in helices 3 and 4. The major surface is topologically similar to the protein interaction surface identified in Drosophila Tube DD and the death effector domain of hamster PEA-15, two physiologically unrelated proteins which interact with structurally unrelated binding partners. These results demonstrate the presence of a structurally conserved surface within the DD which can mediate protein recognition with homo- and heterotypic binding partners, whereas a second surface may be responsible for stabilizing the higher order complex in the DISC.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Role of the integrin-linked kinase/PINCH1/alpha-parvin complex in cardiac myocyte hypertrophy.

Outside-in signaling from fibronectin (FN) through integrin receptors has been shown to play an important role in promoting cardiac myocyte hypertrophy and synergizes with other hypertrophic stimuli such as the alpha-adrenergic agonist phenylephrine (PE) and mechanical strain. The integrin-linked kinase (ILK) is a critical molecule involved in cell adhesion, motility and survival in nonmyocytes such as fibroblasts and epithelial cells. Its role in cardiac myocytes is unclear. In this study, we demonstrate that (1) ILK forms a complex with PINCH1 and alpha-parvin proteins (IPAP1 complex) in neonatal rat ventricular myocytes; (2) localization of IPAP1 complex proteins to costameres in cardiac myocytes is stimulated by FN, PE and synergistically by the combination of FN and PE in an integrin beta1-dependent manner; (3) a dominant-negative mutant lacking the PINCH-binding N-terminus of ILK (ILK-C) prevents costamere association of ILK and alpha-parvin, but not PINCH1; (4) FN- and PE-induced hypertrophy, measured by increased protein/DNA ratio, beating frequency and atrial natriuretic peptide expression, is stimulated by low levels of ILK-C but repressed by high ILK-C expression; and (5) overexpression of ILK-C, as well as deletion of the ILK gene in mouse neonatal ventricular myocytes, induces marked apoptosis of cardiac myocytes. These results suggest that the IPAP1 complex plays an important role in mediating integrin-signaling pathways that regulate cardiac myocyte hypertrophy and resistance to apoptosis.

Actinin↗
PubMed1997

Coupling of the murine protein tyrosine phosphatase PEST to the epidermal growth factor (EGF) receptor through a Src homology 3 (SH3) domain-mediated association with Grb2.

The involvement of murine protein tyrosine phosphatase-PEST (MPTP-PEST) in signal transduction pathways is suggested by its ability to dephosphorylate phosphotyrosine residues, its interaction with the adaptor protein SHC and by the presence of five proline-rich stretches in its non-catalytic carboxyl terminus. Proline-rich sequences have been identified as binding sites for Src homology 3 (SH3) domains found in proteins associated with signal transduction events. The ability of these sequences to act as SH3 domain recognition motifs was investigated using bacterially expressed SH3 domains derived from several different signalling proteins. In vitro binding assays indicate that four of these proline-rich sequences constitute specific binding sites for both SH3 domains of the adaptor molecule Grb2. Wild type Grb2, but not Grb2 proteins corresponding to loss-of-function mutants in the Caenorhabditis elegans sem-5 protein, associate with MPTP-PEST in vivo. Experiments in EGF receptor expressing cells show that the interaction between MPTP-PEST and Grb2 results in the binding of this complex to activated EGF receptors. In addition, identification of putative substrate(s) of MPTP-PEST have revealed a candidate protein of approximately 120 kDa which is tyrosine phosphorylated upon EGF stimulation. Together, these results describe a novel SH3 domain-dependent recruitment of a protein tyrosine phosphatase to an activated receptor tyrosine kinase and establish a potential role for MPTP-PEST in signalling pathways at the molecular level.

3T3 Cells↗
PubMed2004

BCR activation of PI3K is Vav-independent in murine B cells.

BCR (B-cell antigen receptor)-induced Ca(2+) signalling is initiated by activation of tyrosine kinases, which in concert with adaptor proteins and lipid kinases regulate PLC (phospholipase C) gamma2 activation. Vav and PI3K (phosphoinositide 3-kinase) are required for optimal Ca(2+) responses, although it has not been established, in primary B-cells, if both proteins are components of the same pathway. In vitro evidence suggests that binding of the PI3K lipid product PIP3 to Vav pleckstrin homology domain contributes to Vav activation. However, pharmacological inhibition of PI3K by wortmannin or deletion of the p110delta catalytic subunit has no effect on Vav activation in response to BCR engagement, suggesting that this mechanism does not operate in vivo. We also show that PI3K recruitment to phosphorylated-tyrosine-containing complexes is Vav-independent. Taken together with our previous observation that protein kinase B phosphorylation is normal in Vav-deficient B-cells, we suggest that PI3K activation is Vav-independent in response to strong signals delivered by multivalent cross-linking.

Androstadienes↗
PubMed1997

Signal transduction pathway of human fibroblast growth factor receptor 3. Identification of a novel 66-kDa phosphoprotein.

Stimulation of fibroblast growth factor receptor 3 (FGFR3) results in a variety of functional effects, including regulation of epithelial cell growth and differentiation. In order to characterize the signaling pathway through which FGFR3 regulates cell growth, L6 cells lacking any endogenous FGFR were stably transfected with the two different human isoforms, FGFR3 IIIb and FGFR3 IIIc, that result from alternative splicing of exon III of the FGFR3 gene encoding the ligand binding domain. Expression of FGFR3 IIIc in stably transfected L6 cells conferred growth responses to several members of the FGF family including FGF-1, -2, -4, and -6, while FGFR3 IIIb-expressing cells responded only to FGF-1. Activation of FGFR3 upon ligand binding resulted in activation of mitogen-activated protein kinase pathway. FGFR3 utilizes two different pools of adapter protein GRB2 to link to Ras. Activated FGFR3 predominantly interacts with GRB2.Sos in complex with a previously identified 90-kDa protein and designated protein 80K-H. In addition, 80K-H.GRB2. Sos complex was found to contain a novel 66-kDa protein. Tyrosine phophorylation of the 66-kDa protein was dependent on ligand activation of FGFR3, suggesting that the 66-kDa protein may play an important role in FGFR3-specific signaling. In addition to this unique pathway, FGFR3 also links to GRB2.Sos complex via the adapter protein Shc. Furthermore, activated FGFR3 was not able to induce dissociation of GRB2.Sos complex following Sos phosphorylation. In summary, FGFR3 signaling pathway utilizes two GRB2-containing complexes; Shc.GRB2.Sos and 80K-H.pp66.GRB2.Sos; these two complexes may alternatively link FGFG3 to mitogen-activated protein kinase. Finally, activated FGFR3 was also found to result in phosphorylation of phospholipase C-gamma but reduced phosphorylation of c-Src.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Cell signaling: life or death decisions of ras proteins.

The discovery of a new downstream target for the Ras GTPases - a Nore1-Mst1 protein complex - reveals a mechanism by which Ras can promote apoptosis, and suggests that the influence of Ras on cell survival or death depends upon the relative level of activation of its various target proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Regulation of CXCR4-mediated chemotaxis and chemoinvasion of breast cancer cells.

The chemokine-CXCL12 and its receptor, CXCR4, have recently been shown to play an important role in regulating the directional migration of breast cancer cells to sites of metastasis. In the present study, we showed that CXCL12 enhanced the chemotaxis, chemoinvasion and adhesive properties of breast cancer cells; parameters that are critical for development of metastasis. We have also evaluated the signaling mechanisms that regulate CXCL12-induced and CXCR4-mediated breast cancer cell motility and invasion. These studies revealed that CXCL12 induces the tyrosine phosphorylation of focal adhesion kinase (FAK) at residues 397 and 577, and of RAFTK/Pyk2 at residues 402 and 579/580. The cytoskeletal proteins paxillin and Crk, as well as tyrosine phosphatase SHP2 and adaptor protein Cbl, were also phosphorylated. CXCL12 induced the activation of PI 3-kinase, and increased its association with Cbl and SHP2. PI 3-kinase, RAFTK/Pyk2 and tyrosine phosphatase inhibitors significantly blocked CXCL12-induced chemotaxis and chemoinvasion. The role of SHP2 and Cbl in CXCL12-induced chemotaxis and chemoinvasion in breast cancer cells was further defined by transiently overexpressing wild-type SHP2, wild-type Cbl, dominant-negative SHP2, Cbl mutants 70Z/3 and G306E or double transfectants of the Cbl and SHP2 constructs. We found a novel role of Cbl in CXCL12-induced chemotaxis, which may be mediated through the activation and formation of a multimeric complex comprised of Cbl, SHP2 and PI 3-kinase. We also observed the activation of matrix metalloproteinases 2 and 9 upon CXCL12 stimulation. These studies provide new information regarding signaling pathways that may regulate CXCL12-induced metastasis in breast cancer cells.

Breast Neoplasms↗
PubMed2002

Regulation of TSC2 by 14-3-3 binding.

Mutation in either the TSC1 or TSC2 tumor suppressor gene is responsible for the inherited genetic disease of tuberous sclerosis complex. TSC1 and TSC2 form a physical and functional complex to regulate cell growth. Recently, it has been demonstrated that TSC1.TSC2 functions to inhibit ribosomal S6 kinase and negatively regulate cell size. TSC2 is negatively regulated by Akt phosphorylation. Here, we report that TSC2, but not TSC1, associates with 14-3-3 in vivo. Phosphorylation of Ser(1210) in TSC2 is required for its association with 14-3-3. Our data indicate that 14-3-3 association may inhibit the function of TSC2 and represents a possible mechanism of TSC2 regulation.

14-3-3 Proteins↗
PubMed2004

Recruitment of the actin-binding protein HIP-55 to the immunological synapse regulates T cell receptor signaling and endocytosis.

Actin cytoskeleton dynamics critically regulate T cell activation. We found that the cytoplasmic adaptor HIP-55, a Src/Syk-kinases substrate and member of the drebrin/Abp1 family of actin-binding proteins, localized to the T cell-antigen-presenting cell (APC) contact site in an antigen-dependent manner. Using green fluorescent protein fusion proteins, both Src homology 3 (SH3) and actin binding domains were found necessary for recruitment at the T cell-APC interface. HIP-55 was not implicated in conjugate formation and actin polymerization but regulated distal signaling events through binding and activation of hematopoietic progenitor kinase 1 (HPK1), a germinal center kinase (GCK) family kinase involved in negative signaling in T cells. Using RNA interference and overexpression experiments, the HIP-55-HPK1 complex was found to negatively regulate nuclear factor of activated T cell (NFAT) activation by the T cell antigen receptor. Moreover, we show that HIP-55, which partly co-localized with early endocytic compartments, promoted both basal and ligand-dependent T cell receptor (TCR) down-modulation, resulting in a decreased TCR expression. SH3 and actin-depolymerizing factor homology domains were required for this function. As controls, the expression of CD28 and the glycosylphosphatidylinositol-linked protein CD59 was not affected by HIP-55 overexpression. These results suggest that, in addition to binding to HPK1, HIP-55 might negatively regulate TCR signaling through down-regulation of TCR expression. Our findings show that HIP-55 is a key novel component of the immunological synapse that modulates T cell activation by connecting actin cytoskeleton and TCRs to gene activation and endocytic processes.

Actins↗